Previously we found that intra-ventral tegmental injections of the cholinergic agonist carbachol produce a rewarding effect; rats self-administer carbachol at a relatively fast rate directly into the ventral tegmental area (VTA). We sought to determine brain regions that are activated by intra-VTA carbachol using the immunohistochemical technique for detection of c-Fos protein. Nine brain regions were examined to determine the number of neurons expressing c-Fos immunoreactivity in 13 rats: 5 rats received 500 pmol carbachol into the posterior VTA, 5 received the carbachol solution into the region 1 mm dorsal to the VTA, and 3 received vehicle (0.5 ?l) into the VTA. We observed notable increase (>400%) of c-Fos expression in four brain areas after intra-VTA carbachol injections: retrosplenial granular (posterior cingulate) cortex (500%), supramammillary nucleus (700%), anterior VTA (400%), and interpeduncular nucleus (1700%). Furthermore, locomotor distance during the first 10 min just after microinjections was significantly correlated with c-Fos expression in the retrosplenial granular cortex (0.71), supramammillary nucleus (0.70), and anterior VTA (0.73), but not the posterior VTA (0.12; injection site) or interpeduncular nucleus (0.45; most carbachol-sensitive site with respect to % increase from vehicle baseline). The high correlation coefficient between carbachol-induced c-Fos activation and locomotor activation suggest that in particular, retrosplenial granular cortex, supramammillary nucleus, and anterior VTA play an important role in intra-VTA carbachol induced locomotion, and possibly reward. Whether or not carbachol is exciting or inhibiting those neurons that initiate the behavioral effects remains to be determined.